This invention relates generally to chromatography systems, and more specifically relates to liquid chromatography systems.
Chromatography is a separation method wherein a mixture of components (called the "sample" or "sample mixture") is placed as a zone at one end of a system containing a stationery phase and a mobile phase. Each component of the sample distributes itself in dynamic equilibrium between the two phases, in a ratio characteristic of that component. As a result, the flowing mobile phase causes each individual component zone to migrate at a characteristic rate, and the zones become separated after a period of time. There are various types of chromatography, e.g., liquid chromatography, gas chromatography, thin-layer chromatography, etc. The major difference between these various chromatographic methods is the physical state of the mobile phase (gas or liquid) and the manner in which the stationary phase is supported (e.g., coated on an inert granular material packed in a tube, coated on an inert plate, etc.). In each method, the separation mechanism is essentially the same, i.e., distribution of the sample components between the mobile phase and a stationary phase. When the method is used for chemical analysis, a detector is often placed at the other end of the system, so as to monitor the passage of the component zones as they emerge from the system. The signal from the detector is displayed on a recording device such as a strip chart recorder, and the record indicates both qualitative and quantitative information regarding the components of the sample.
It is often desirable for a chromatographic system to provide high resolution (i.e., a large degree of component separation with narrow zones), evenly spaced component zones, rapid separation, and a satisfactory record from a very small sample. The behavior of the system described in these terms may be called the "performance" of the system. It is well-known in the chromatography art to improve system performance by changing one of the following system variables during the course of the analysis: temperature, chemical composition of the mobile phase, and flow rate of the mobile phase. For example, in gas chromatography the temperature of the system is often varied as a pre-selected function of time. This technique is known as "temperature programming", and it improves the performance of the system, especially with samples containing components which boil over a wide temperature range.
Analagous to temperature programming in gas chromatography is the use of "gradient elution" in liquid chromatography. Gradient elution refers to changing the chemical composition of the mobile phase (also called the "eluent" or "eluting solvent") as a function of time, thereby improving the performance of the system, especially with samples containing components which vary widely in chemical properties. A further example of changing the chromatographic variables is the recent development of "flow programming" in gas and liquid chromatography, wherein the flow rate of the mobile phase is changed as a pre-selected function of time. As mentioned previously, the object of changing or "programming" the individual chromatographic system variables during the analysis is to improve one or more aspects of system performance. Further discussion with regard to gradient elution techniques and the factors affecting system performance may be found in various places in the art, including, e.g., U.S. Pat. No. 3,446,057, and the publication by L. R. Snyder appearing in Chromatography Review 7, 1 (1965).
The normal and usual arrangement in chromatography apparatus of the type considered herein entails use of one or more reservoirs, which are basically in the nature of syringe pumps. A given said reservoir thus may comprise a cylindrical tube or the like, having a volume V. A piston of circular cross-section is mounted for axially-directed movement in the cylinder, and is normally driven by motor means at a pre-selected velocity which may be constant over a given period of time, or which varies in accordance with the gradient elution program.
It has been found that a most serious technical problem arising in the use of apparatus as mentioned above derives from a failure to account for compressibility of the several solvents. In particular, during the course of gradient elution work, viscosity changes occur in the composite liquid phase flowing through the chromatography column. This, in turn, induces pressure changes at the input of the said column, i.e., in the flow path between the reservoirs and the said column. By virture of the compressibility of the various solvents, these pressure changes, interacting with the already programmed velocities of the pistons, induce density changes in the solvents, which is to say, changes in the volume of a given mass of the said solvents. The net effect of these changes, which, of course, arise by virture of compressibility of the solvents, is to effectively change the flow rates of one or more of the solvents-- with possibly highly detrimental effects on system performance.
The phenomenon in turn, under such conditions, may be further appreciated by considering that the pressure drop across the chromatographic column is represented by the simple expression: EQU P= VR (1)
where P is the pressure drop or pressure in the pump, V is the volumetric flow rate, and R is the column resistance in appropriate units. The resistance R is proportional to viscosity for laminar flow-- which normally prevails in liquid chromatography. As the viscosity changes, so does the column resistance. Since the pump is pre-programmed to have a certain piston velocity movement, which over a given time period maintains a constant volumetric flow rate, the pressure in the pump increases with increase in viscosity of the fluid. This increase in pressure causes compression of the liquid in the pump (or pumps in the gradient system). This compression subtracts from the output flow rate of the pump, so that the flow rate is no longer constant. This effect can be dramatic when the pump reservoirs are nearly full, and the change in solvent composition is rapid compared to the output flow rate of the pumping system. Thus it may readily be shown that in a gradient system based on water and methanol, wherein the gradient operation is such as to proceed from water to 40% methanol in water over a period of 10 minutes, and wherein the pressure with water is 2500 psi, the nominal flow rate is 60 ml/hr, and the volume of each reservoir is 200 cc, a 40% deviation of actual flow from the nominally set flow rate can ensue.
One conceivable solution to the foregoing difficulty is to provide each pump in the gradient system with a flow rate measuring device, and feed back the response from such device to maintain a constant or other prescribed flow rate function from the pump. The complexity of each arrangement however introduces inordinate cost and complexity into a pumping system.
In accordance with the foregoing, it may be regarded as an object of the present invention to provide means enabling maintenance of a constant flow in a liquid chromatography system in the presence of flow resistance changes at the chromatographic column of such system, which changes are induced by viscosity changes in the liquid phase deriving from gradient elution techniques. Changes in viscosity of lesser magnitude can arise from temperature variations. Column resistance can also change with change in column bed structure such as the phenomena usually referred to as bed settling.
It is a further object of the present invention to provide means enabling constant flow through the chromatographic column in a liquid chromatography system of the type considered in the preceding paragraph, wherein, further, the said means is of great simplicity thereby assuring ease of operation and low cost for construction thereof.